The present invention relates to novel complexes of platelet-derived growth factor (PDGF) associated with amphiphilic polymers for improving the physical and chemical stability, in vitro and in vivo, of the therapeutic protein for pharmaceutical applications.
PDGFs are glycoproteins of approximately 30 000 daltons, made up of two polypeptide chains linked to one another via two disulphide bridges. Four types of chains have been identified, A, B, C and D. The native protein exists in the form of a homodimer or of an AB-type heterodimer (Oefner C. EMBO J. 11, 3921-2926, 1992).
PDGF was isolated for the first time from platelets. PDGFs are growth factors released during blood clotting, capable of promoting the growth of various cell types (Ross R. et al., Proc. Natl. Acad. Sci. USA, 1974, 71, 1207; Kohler N. & Lipton A., Exp. Cell Res., 1974, 87, 297). It is known that PDGF is produced by a certain number of cells other than platelets, and that it is mitogenic with respect to most of the cells derived from the mesenchyma, i.e. blood, muscle, bone and cartilaginous cells, and also connective tissue cells (Raines E. W., in “Biology of Platelet-Derivated Growth Factor”, 1993, Westermark, B. and C. Sorg, Ed. Basel, Kerger, p. 74). Many articles tend also to demonstrate that macrophage-derived PDGF behaves like a chemotactic and mitogenic agent with respect to smooth muscle cells, and that it contributes to the myointimal thickening of arterial walls characteristic of arteriosclerosis (Ross R. et al., Science, 1990, 248, 1009). The activities of PDGF include, in addition and in particular, the stimulation of granule release by neutrophilic monocytes (Tzeng D. Y. et al., Blood, 1985, 66, 179), the facilitation of steroid synthesis by Leydig cells (Risbridger G. P., Mol. Cell, Endocrinol., 1993, 97, 125), the stimulation of neutrophil phagocytosis (Wilson E. et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 2213), the modulation of thrombospondin expression and secretion (Majak R. A. et al., J. Biol. Chem., 1987, 262, 8821), and the post-regulation of the ICAM-1 gene in vascular smooth muscle cells (Morisaki N. et al., Biochem. Biophys. Res. Commun., 1994, 200, 612).
Given these various properties, the use of recombinant PDGFs in the pharmaceutical field has already been envisaged. The use of PDGF has in particular been approved for the treatment of diabetic foot ulcers (Regranex, J&J) and for periodontal repair (GEM 21S, Biomimetic).
Ulcer healing, just like cutaneous healing in general, is a complex phenomenon that requires the coordinated intervention, over time and in space, of numerous cell types, that can be summarized in three phases: an inflammation phase, a proliferation phase and a remodeling phase.
In the inflammation phase, which is approximately 7 days for normal healing, macrophages kill the bacteria, debride damaged tissues and regenerate the tissues. To do this, the macrophages secrete collagenases, cytokines and growth factors.
In the course of the proliferation phase, which is from the 3rd day to the 3rd week for normal healing, three events follow on from one another. The wound fills with granulation tissue, angiogenesis develops and the wound becomes covered with epithelial cells. The granulation tissue grows from the edges to the centre. Fibroblasts abundantly produce collagen type III.
In the course of the remodeling, which is from the 3rd week to 1 or even 2 years, the granulation tissues mature, and the fibroblasts produce less collagen. The blood vessels formed during the granulation that are of no use are eliminated by apoptosis. The collagen type III is replaced with collagen type I, which organizes according to lines of tension and crosslinks.
In this process, PDGF plays an essential role. During the formation of a wound, platelets aggregate and release PDGF. The PDGF attracts neutrophils, macrophages and fibroblasts to the wound and is a potent mitogen. The macrophages and the endothelial cells in turn synthesize and secrete PDGF. The PDGF stimulates the production of the new extracellular matrix by the fibroblasts, essentially the non-collagen compounds such as glycosaminoglycans and the adhesion proteins (J. F. Norton et al, Essential practice of surgery, Springer, 2003, chapter 7, 77-89).
Chronic wounds such as diabetic foot ulcers, venous ulcers and pressure ulcers have the particularity of healing very slowly and sometimes incompletely because the healing process does not take place normally (R. Lobmann et al., J. of Diabetes and its complications, 2006, 20, 329-335).
The healing process is in fact a delicate balance between a process of destruction necessary in order to eliminate the damage to tissues and the repair process that results in the formation of new tissues. Proteases and growth factors play an essential role in this process by regulating this balance. In the case of chronic wounds, this balance is disturbed in favour of degradation, therefore these wounds are slow to heal. Although different types of chronic wounds exist, they are biochemically relatively similar in the sense that they are characterized by sustained inflammation phases that result in high levels of proteases and thus decrease growth factor activity (G. Lauer et al. J. Invest. Dermatol. 115 (2000) 12-18). This growth factor degradation contributes to an overall loss of tissues associated with these chronic wounds that does not favour healing (D. R. Yager et al., J. Invest. Dermatol. 107 (1996) 743-748).
There currently exists on the market a human recombinant PDGF-BB-based medicament corresponding to the international non-proprietory name “becaplermin”, sold under the trade name Regranex®. This medicament is indicated for the treatment of lower limb ulcers in diabetics. It is in the form of a gel for topical application and makes it possible to promote ulcer healing. It makes it possible in particular, just like endogenous PDGF, to promote cell proliferation and therefore the formation of new tissues.
This treatment has a limited efficacy (Cullen et al. The international journal of biochemistry & Cell Biology 34, 1544-1556, 2002) even though the clinical studies have shown improvements in healing and in the period of time required for healing (Greenhalgh et al., American Journal of pathology, 136, 1235-1246 1990; Ladin Plastic and Reconstructive Surgery, 105, 1230-1231 2000; Holloway et al. Wounds, 5/4, 198-206; Mandracchia et al. Clinics in Podiatric Medicine and Surgery, 18, 189-209 2001; Wieman T. J. American Journal of Surgery, 176, 74S-79S 1998).
The Regranex product that contains PDGF-BB, sold by J&J, has demonstrated its efficacy by increasing the rate of recovery, in the patients treated, to 50% against only 36% for patients who only received standard treatment of the wound. Despite this significant improvement in the treatment of diabetic foot ulcers, it must be noted that only 50% of the patients experience recovery after a long and expensive treatment. In the cases of non-recovery, the consequences can be extremely serious and, in many cases, result in amputation of the lower limb. It should be added that the average duration of the treatment is very long, approximately 20 weeks, and the application thereof is expensive and restrictive because the wound must be cleaned and Regranex applied in the morning, followed 12 hours later, by cleaning of the wound. These two procedures most commonly require care by a nurse. In addition, the average cost of a treatment lasting twenty weeks is excessively high (of the order of 1400 American dollars).
The partial efficacy can be explained by a rapid degradation of the PDGF on the wound to be treated. This degradation results, in the case of a chronic wound, from a sustained inflammation state generating, at the wound, an environment hostile to the PDGF due to the stimulation of an overproduction of proteases.
Although degradation control is necessary for wound healing, excessive proteolytic activity is harmful since it leads to degradation of the extracellular matrix (F. Grinnell et al. J. Invest. Dermatol. 106 (1996) 335-341 and C. N. Rao et al. J. Invest. Dermatol. 105 (1995) 572-578) and of molecules that have a key functional role such as growth factors (V. Falanga et al. J. Derm. Surg. One. 18 (1992) 604-606; D. R. Yager et al. Wound Rep, Reg. 5 (1997) 23-32. and M. Wlaschek et al. Br. J. Dermatol. 137 (1997) 646-647). In fact, growth factors such as PDGF, TGFβ or bFGF are key elements in the healing process due to their abilities to induce cell migration, proliferation, protein synthesis and matrix formation and, more generally, due to the fact that they control the repair process. However, these growth factors are protein molecules and, consequently, are sensitive to proteolytic degradation. Several studies show that the degradation of growth factors such as PDGF is much more rapid when they are brought into contact with fluids originating from chronic wounds since they contain high concentrations of metalloproteinases (D. R. Yager et al. J. Invest. Dermatol. 107 (1996) 743-748).
For the treatment of venous ulcers, Regranex, in a pilot clinical study reported in the publication (T. J. Wieman, Wounds, 2003, vol. 15, No. 8, 257-264), showed only a minor improvement of current treatments based on regular cleaning of the wound with compression therapy.
The problem of the instability of PDGF, for example, was revealed during the production of the protein. It is known that PDGF is particularly sensitive to post-translational proteolysis (Hart et al., Biochemistry 29:166-172, 1990 and U.S. patent Ser. No. 07/557,219) and especially at the level of the bond between the arginine amino acid at position 32 and the threonine amino acid at position 33 of the mature chain of the protein. Other sites are sensitive to proteolysis, such as the bond between the arginine at position 79 and the lysine at position 80, or else the bond between the arginine at position 27 and the arginine at position 28 of the B chain of PDGF.
This proteolytic instability poses a major problem in the context of obtaining this protein, which is produced in a recombinant manner in yeast according to the method described in U.S. Pat. No. 4,845,075. Specifically, U.S. Pat. No. 7,084,262 teaches us that the analysis and purification of PDGF-BB leads to the production of 21 isoforms resulting from post-translational endoproteolytic cleavages. This great structural heterogeneity consequently results in a 50% decrease in the activity of the protein produced by genetic engineering compared with the intact protein in mature form.
Moreover, recent clinical results by Cardium, according to a press communiqué dated 14 Aug. 2006 (www.prnewswire.com) on diabetic foot ulcers that have not recovered after 14 weeks, shows the potential that a treatment with PDGF-BB can offer. The solution proposed by Cardium consists in introducing the gene for expression of PDGF-BB into the cells of the wound so as to overexpress it locally. This gene therapy by means of an adenovector made it possible to heal close to 80% of these diabetic foot ulcers resistant to the common treatments, out of a group of 15 patients. This therapeutic solution is promising. However, pharmaceutical developments of gene therapy-based treatments are, to date, still very hazardous for reasons of safety linked to the use of adenovirus-type viral vectors.
There is therefore a need and the possibility to improve the current treatments for diabetic foot ulcers, with PDGF.
In the case of diabetic foot ulcer treatment, the final objective is three-fold:                to accelerate recovery        to increase the rate of recovery        to simplify the treatment protocol.        
There is also the case of venous ulcers and of pressure ulcers, which are the cause of considerable pain and of very serious medical complications.
The problem to be solved is therefore essentially that of protecting PDGF on chronic wounds.
Various solutions have been proposed.
U.S. Pat. No. 5,905,142 describes a means of remedying these proteolysis problems concerning PDGF by generating mutants of the protein that have an increased resistance with respect to proteolytic attacks, by substituting or by deleting one or more lysine or arginine amino acids close to the potential cleavage sites. This strategy for making the protein more resistant to proteases is not satisfactory. This genetic modification of PDGF can lead to modifications of the biological activity, with affinities that are different with respect to its various receptors, which can also induce toxicological problems. In addition, such a modification of PDGF requires a new pharmaceutical development, which is extremely expensive and risky.
In the 1970s, when this protein was abundantly studied, it was found that purification was extremely tricky since PDGF is “a very sticky protein” due to its cationic and hydrophobic properties (Heldin, C. H. EMBO J. 11:4251-4259, 1992; Raines and Ross, J. Biol. Chem. 257(9):5154-5160, 1982; Antoniades, PNAS 78:7314, 1981; Deuel et al. J. Biol. Chem. 256:8896, 1981). PDGF is in fact a highly cationic protein, the isoelectric point of which is between 9.8 and 10.5. Other authors confirm this behaviour, such as Wei et al. (Journal of controlled release 112:103-110, 2006), who explain that PDGF readily adsorbs onto surfaces of the container in which it is in solution. The authors solve the problem by adding, to the mixture, either 0.1% BSA or a 0.1% BSA/Tween 20 mixture. These solutions solve the problems to a large extent since up to 95% of the protein is found in solution. However, these solutions are not satisfactory from a pharmaceutical point of view, given the animal origin of the BSA and the risks related to bovine spongiform encephalopathy.
Another solution proposed by the same authors consists in adding a more powerful anionic surfactant (SDS), that makes it possible to maintain the PDGF in solution. Unfortunately, SDS also induces partial denaturation of the protein, resulting in a loss of bioactivity. This solution is not therefore satisfactory for stabilizing the protein.
In patent WO 93/08825, the inventors have demonstrated that purified PDGF exhibits great instability when it is formulated in the form of a gel for topical application. They give, as an example, the incompatibility of PDGF with a certain number of products conventionally used for formulating pharmaceutical products, such as methylcellulose or hydroxypropylcellulose, and also certain conventional preserving agents such as benzyl alcohol. The authors pose the problem by explaining that there exists a need to formulate PDGF in the form of a gel for topical administration while at the same time having good long-term stability. The same authors show that PDGF in solution degrades due to a process of deamidation at neutral pH and that the protein is more stable at a slightly acidic pH. The authors show that, by combining several parameters, a polymer that does not exhibit any interactions with the protein, a buffer at slightly acidic pH making it possible to limit the deamidation reaction and a preserving agent that is neutral with respect to the protein, it is possible to formulate the PDGF in order to obtain a formulation that is stable from a pharmaceutical point of view.
The authors show that it is possible to obtain a storage-stable formulation through the addition of a polymer that does not exhibit any interactions with the protein, with the proviso that the formulation is maintained at a slightly acidic pH in order to avoid reactions that degrade the protein by deamidation. This solution is not, however, satisfactory since it does not make it possible to protect the growth factor against proteolytic degradations in vivo at physiological pH.
In patent WO 97/12601, which describes PDGF formulations in the form of a gel, the authors explain that the cellulose derivative that they use is capable of stabilizing growth factors against a possible loss of activity during storage. For this, they use the results obtained previously on EGF in U.S. Pat. No. 4,717,717 as a basis. However, they also explain that the stability of the cellulose gel containing PDGF can be greatly improved by adding to the formulation a charged chemical species such as charged amino acids or metal ions. Here again, this solution makes it possible to stabilize the growth factors in the formulation during the storage of the product, but does not allow a stabilization of these growth factors with respect to the proteases present in chronic wounds at physiological pH.